Field of the Invention
The present invention relates to an improved microorganism for the production of methionine and process for the preparation of methionine. In particular, the present invention relates to a microorganism for methionine production with improved glucose import comprising modified expression of at least one gene selected from ptsG, sgrS, sgrT or dgsA.
Description of Related Art
Sulphur-containing compounds such as cysteine, homocysteine, methionine or S-adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless, most of the methionine that is produced is added to animal feed.
With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Commonly, D,L-methionine is produced chemically from acrolein, methyl mercaptan and hydrogen cyanide. However, the racemic mixture does not perform as well as pure L-methionine (Saunderson, C. L., 1985). Additionally, although pure L-methionine can be produced from racemic methionine, for example, through the acylase treatment of N-acetyl-D,L-methionine, this dramatically increases production costs. Accordingly, the increasing demand for pure L-methionine coupled with environmental concerns render microbial production of methionine an attractive prospect.
Optimising the production of a chemical from a microorganism typically involves overexpressing proteins involved in the biosynthesis pathway, attenuating proteins involved in repression of the biosynthesis pathway or attenuating proteins involved in the production of undesirable by-products. All these approaches for the optimisation of L-methionine production in microorganisms have been described previously (see, for example, U.S. Pat. No. 7,790,424, U.S. Pat. No. 7,611,873, patent applications WO 2002/10209, WO 2006/008097 and WO 2005/059093); however, industrial production of L-methionine from microorganisms requires further improvements.
Typically, L-methionine has been produced by microorganisms grown on glucose as a main carbon source in a fermentative process. In bacteria, external glucose is transported into the cell and phosphorylated by the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Meadow et al. 1990; Rohwer et al. 1996; Tchieu et al. 2001). The PTS consists of two common cytoplasmic proteins, enzyme I and HPr, and a series of sugar-specific enzyme II complexes (EIIs). The PTS enzyme IICBGlc, encoded by ptsG in E. coli transports and concomitantly phosphorylates glucose to glucose-6-phosphate (G6P). While G6P is an essential intermediate in glucose metabolism, its intracellular accumulation causes the phenomenon of sugar-phosphate toxicity, also called “phosphosugar stress”. Indeed, the accumulation of glucose has been reported to be very toxic for bacteria, giving rise to glycation, DNA mutagenesis and growth inhibition (Lee and Cerami, 1987; Kadner et al. 1992).
Recent studies have demonstrated that in E. coli the ptsG gene encoding IICBGlc is highly regulated in quite an intriguing manner at both the transcriptional and post-transcriptional level, depending on physiological conditions (Plumbridge, 1998; Kimata et al. 1998; Plumbridge et al. 2002; Morita and Aïba, 2007; Görke and Vogel, 2008). Specifically, several levels of regulation have been identified:
regulation of the expression of ptsG gene by many different regulators (ArcA, Fis, Crp) and in particular the repression by DgsA, a transcriptional regulator firstly called Mlc (Making larger colonies);
destabilization of the ptsG mRNA by the small RNA sgrS (Sugar transport-related sRNA) by an antisense mechanism;
control of PtsG activity by the small polypeptide SgrT by a yet unknown mechanism; and
regulation of the expression of sgrS/sgrT by the transcriptional regulator SgrR.
Due to the toxicity of G6P and the highly regulated and complex nature of the system, manipulation of the glucose transport system in microorganisms is very difficult. To date there have been several attempts to improve amino acids production and in particular threonine production by increasing glucose import by manipulating ptsG or dgsA genes (WO03004675 and WO03004670 of Degussa; US2004229320 of Ajinomoto and WO0281721 of Degussa). Nevertheless, there is no example of improving the production of methionine by increasing the glucose import of the bacterium.